One of the most exciting revelations in retinal biology is that the molecules and mechanisms that direct eye formation are conserved throughout the animal kingdom. In recent years an entire cascade of molecules and their roles in eye specification have been found to be preserved in both mammalian and invertebrate retinal systems. In addition to provoking a profound rethinking of the origins of the eye, the emerging commonality among mechanisms used in eye development allows for the use of model systems to provide insight into the development and diseases of the mammalian eye. The Sine oculis box (Six) and Eyes absent (Eya) gene families function within the signaling cascades that regulate eye and head development in both mammals and invertebrates. Bilateral anophthalmia and holoprosencephaly type 2 are caused by mutations within the human Six6 and Six3 genes, while molecular lesions within the human Eyal gene result in congenital cataracts, Branchio-Oto-Renal (BOR) and Oto-FaciaI-Cervial (OFC) syndromes. The association of Six and Eya gene lesions with eye diseases, provides us with an exciting opportunity to explore the roles played by the SIX and EYA proteins in retinogenesis. The developing eye of the fruit fly, Drosophila melanogaster, is an excellent experimental system for elucidating the genetic, molecular and biochemical mechanisms underlying the activities of the SIX and EYA proteins. In the fruit fly, the Six family is represented by the SINE OCULIS (SO) and OPTIX proteins, while the EYES ABSENT (EYA) protein is the only Eya family member. The goals of the proposed work are (1) to identify the steps in eye specification that are regulated by the Drosophila SIX and EYA family members; (2) to define the functional differences between the two fruit fly SIX proteins; (3) to understand the interactions of several signaling pathways with SO; and (4) to identify additional genes whose products participate in eye development and interact with SO. To achieve these goals, eye development will be analyzed in animals lacking both SO and EYA proteins; biochemical approaches will be used to dissect functional differences between SO and OPTIX; and the well-established techniques of Drosophila genetics will be used to define relationships between SO and other key proteins involved in eye development. Together, these approaches will provide insight into the roles of the SIX and EYA proteins in eye specification.